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Nucleocapsid formation and RNA synthesis of Marburg virus is dependent on two coiled coil motifs in the nucleoprotein.

DiCarlo A, Möller P, Lander A, Kolesnikova L, Becker S - Virol. J. (2007)

Bottom Line: In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor.Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system.The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker.

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Affiliation: Philipps-Universität Marburg, Institut für Virologie, Hans Meerwein-Str, 2, 35032 Marburg, Germany. andrea.dicarlo@promega.com

ABSTRACT
The nucleoprotein (NP) of Marburg virus (MARV) is responsible for the encapsidation of viral genomic RNA and the formation of the helical nucleocapsid precursors that accumulate in intracellular inclusions in infected cells. To form the large helical MARV nucleocapsid, NP needs to interact with itself and the viral proteins VP30, VP35 and L, which are also part of the MARV nucleocapsid. In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor. Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system. Transfer of the coiled coil motif to a reporter protein was sufficient to mediate interaction of the constructed fusion protein with the N-terminus of NP. The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker.

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Role of the coiled coil region for interaction of NP with VP35. (A) Mutants of NP (Fig. 1) were in vitro translated and metabolically labeled with [35S]ProMix. Samples were separated by SDS-PAGE and analyzed using a BioImager. (B) In vitro translated and [35S]ProMix metabolically labeled mutants of NP (Fig. 1A) were incubated with bacterially expressed GST-VP35 or GST (negative control). Complexes were pulled down with glutathion-sepharose, separated on SDS-PAGE and analyzed using a BioImager. Binding of NP to GST-VP35 was set to 100%. (C) Quantification of 3 separate experiments as shown under (B).
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Figure 3: Role of the coiled coil region for interaction of NP with VP35. (A) Mutants of NP (Fig. 1) were in vitro translated and metabolically labeled with [35S]ProMix. Samples were separated by SDS-PAGE and analyzed using a BioImager. (B) In vitro translated and [35S]ProMix metabolically labeled mutants of NP (Fig. 1A) were incubated with bacterially expressed GST-VP35 or GST (negative control). Complexes were pulled down with glutathion-sepharose, separated on SDS-PAGE and analyzed using a BioImager. Binding of NP to GST-VP35 was set to 100%. (C) Quantification of 3 separate experiments as shown under (B).

Mentions: We then investigated whether the integrity of the coiled coil region of NP is important for the binding of NP to the polymerase cofactor VP35, which connects the polymerase L to the NP-induced helical nucleocapsid [6,10]. The coding region of VP35 was fused to GST and the fusion protein (GST-VP35) was expressed in E. Coli and, following purification, was incubated with in vitro translated NP or NP mutants containing deletions of the coiled coil motifs (Fig. 3A). In a GST pull-down assay, GST-VP35 was pulled down by glutathione-sepharose and the amount of coprecipitated NP and mutants of NP was quantified to assess the interaction of VP35 with the coiled coils (Figs. 3B and 3C). While NP was readily precipitated by GST-VP35, deletion of C1 significantly decreased the amount of coprecipitated protein (Figs. 3B and 3C, ΔC1). Deletion of C2 seemed to increase the binding of GST-VP35 to the NP mutant (Figs 3B and 3C, ΔC2). These data indicate that the coiled coil region of NP influences the interaction with VP35.


Nucleocapsid formation and RNA synthesis of Marburg virus is dependent on two coiled coil motifs in the nucleoprotein.

DiCarlo A, Möller P, Lander A, Kolesnikova L, Becker S - Virol. J. (2007)

Role of the coiled coil region for interaction of NP with VP35. (A) Mutants of NP (Fig. 1) were in vitro translated and metabolically labeled with [35S]ProMix. Samples were separated by SDS-PAGE and analyzed using a BioImager. (B) In vitro translated and [35S]ProMix metabolically labeled mutants of NP (Fig. 1A) were incubated with bacterially expressed GST-VP35 or GST (negative control). Complexes were pulled down with glutathion-sepharose, separated on SDS-PAGE and analyzed using a BioImager. Binding of NP to GST-VP35 was set to 100%. (C) Quantification of 3 separate experiments as shown under (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2170442&req=5

Figure 3: Role of the coiled coil region for interaction of NP with VP35. (A) Mutants of NP (Fig. 1) were in vitro translated and metabolically labeled with [35S]ProMix. Samples were separated by SDS-PAGE and analyzed using a BioImager. (B) In vitro translated and [35S]ProMix metabolically labeled mutants of NP (Fig. 1A) were incubated with bacterially expressed GST-VP35 or GST (negative control). Complexes were pulled down with glutathion-sepharose, separated on SDS-PAGE and analyzed using a BioImager. Binding of NP to GST-VP35 was set to 100%. (C) Quantification of 3 separate experiments as shown under (B).
Mentions: We then investigated whether the integrity of the coiled coil region of NP is important for the binding of NP to the polymerase cofactor VP35, which connects the polymerase L to the NP-induced helical nucleocapsid [6,10]. The coding region of VP35 was fused to GST and the fusion protein (GST-VP35) was expressed in E. Coli and, following purification, was incubated with in vitro translated NP or NP mutants containing deletions of the coiled coil motifs (Fig. 3A). In a GST pull-down assay, GST-VP35 was pulled down by glutathione-sepharose and the amount of coprecipitated NP and mutants of NP was quantified to assess the interaction of VP35 with the coiled coils (Figs. 3B and 3C). While NP was readily precipitated by GST-VP35, deletion of C1 significantly decreased the amount of coprecipitated protein (Figs. 3B and 3C, ΔC1). Deletion of C2 seemed to increase the binding of GST-VP35 to the NP mutant (Figs 3B and 3C, ΔC2). These data indicate that the coiled coil region of NP influences the interaction with VP35.

Bottom Line: In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor.Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system.The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker.

View Article: PubMed Central - HTML - PubMed

Affiliation: Philipps-Universität Marburg, Institut für Virologie, Hans Meerwein-Str, 2, 35032 Marburg, Germany. andrea.dicarlo@promega.com

ABSTRACT
The nucleoprotein (NP) of Marburg virus (MARV) is responsible for the encapsidation of viral genomic RNA and the formation of the helical nucleocapsid precursors that accumulate in intracellular inclusions in infected cells. To form the large helical MARV nucleocapsid, NP needs to interact with itself and the viral proteins VP30, VP35 and L, which are also part of the MARV nucleocapsid. In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor. Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system. Transfer of the coiled coil motif to a reporter protein was sufficient to mediate interaction of the constructed fusion protein with the N-terminus of NP. The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker.

Show MeSH
Related in: MedlinePlus